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1.
Environ Technol ; : 1-13, 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30379618

RESUMO

A series of MnOx-CeO2 catalysts with MOx doping (M = Cu, Fe, Co and La) supported on cordierite were synthesized by the citric acid complex method, showing preferable catalytic oxidation of chlorobenzene. The distribution of active oxides, surface areas, as well as the structural morphology of M-MnOx-CeO2 catalysts varied with the different Mn/Ce and M/Mn molar ratios. Meanwhile, physicochemical properties of these catalysts were characterized by XRD, BET, SEM, TEM, H2-TPR and IR. More importantly, the catalytic oxidation routes were also investigated where the process was from chlorobenzene to CO2, H2O, HCl and other by-products for the FeOx-MnOx-CeO2 and CuO-MnOx-CeO2 catalysts. The CuO-MnOx-CeO2 catalysts showed a higher chlorobenzene conversion, and the measured light-off temperature T90 was approximately 400°C. However, a large amount of chloropropane as main by-products was observed. For the FeOx-MnOx-CeO2 catalysts, more carbon monoxide could be found with inadequate oxidation. Comparative analyses of two catalysts indicated that the main cause of the oxidation activities and mechanisms were different in the oxidation capacity and water absorbability of FeOx and CuO. Nevertheless, all of these catalysts did not exhibit any deactivation due to chloride with a high reaction temperature, with chloride transformed to form HCl in the off-gas stream.

2.
Protein Sci ; 24(12): 2044-54, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26440996

RESUMO

The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed ß-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.


Assuntos
Álcool Desidrogenase/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Álcool Desidrogenase/metabolismo , Asparagina/metabolismo , Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Modelos Moleculares , Polietilenoglicóis/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
3.
Biol Trace Elem Res ; 168(1): 103-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25860427

RESUMO

This study was conducted to evaluate the efficacy of a novel Buttiauxella phytase to pigs fed P-deficient, corn-soybean meal diets. One hundred and twenty crossbred piglets (9.53 ± 0.84 kg) were allocated to one of five treatments which consisted of four low P diets (0.61 % Ca and 0.46 % total P) supplemented with 0, 500, 1,000, or 20,000 FTU/kg phytase as well as a positive control diet (0.77 % Ca and 0.62 % total P). Each treatment had six replicated pens with four pigs per pen. Pigs were fed the experimental diets for 28 days. Phytase supplementation linearly improved (P < 0.05) average daily gain (ADG), feed conversion ratio (FCR), and apparent total tract digestibility (ATTD) of dry matter, gross energy, crude protein, Ca, and P in weaned pigs. Super high dosing with phytase (20,000 FTU/kg) further increased (P < 0.05) ADG compared with 500 FTU/kg phytase inclusion group, as well as ATTD of Ca and P. Metacarpal bone characteristics and several trace mineral concentration in bone, plasma, or organ tissues were linearly (P < 0.05) improved at increasing dose of phytase. Super high dosing with phytase (20,000 FTU/kg) supplementation improved (P < 0.05) Mn and Zn concentration in bone compared to normal dose of phytase supplementation (500 or 1,000 FTU/kg). In conclusion, supplementation of 500 FTU of Buttiauxella phytase/kg and above effectively hydrolyzed phytate in a low-P corn-soybean diet for pigs. In addition, a super high dosing with phytase (20,000 FTU/kg) improved macro- or micro mineral availability and growth performance.


Assuntos
6-Fitase/farmacologia , Digestão/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Crescimento/efeitos dos fármacos , Minerais/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Densidade Óssea , Desenvolvimento Ósseo/efeitos dos fármacos , Suplementos Nutricionais , Estado Nutricional/efeitos dos fármacos , Fósforo/deficiência , Soja , Sus scrofa , Suínos , Zea mays
4.
J Agric Food Chem ; 63(7): 2006-12, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25660114

RESUMO

Ferulic acid dehydrodimers (DFA) and dehydrotrimers (TriFA) ester-linked to plant cell wall polymers may cross-link not only cell wall polysaccharides but also other cell wall components including proteins and lignin, thus enhancing the rigidity and potentially affecting the enzymatic degradation of the plant cell wall. Corn, wheat, and mixed-cereal distiller's dried grains with solubles (DDGS) were investigated for composition of DFAs and TriFAs by reversed phase high-performance liquid chromatography with ultraviolet detection. Corn DDGS contained 5.3 and 5.9 times higher contents of total DFAs than wheat and mixed-cereal DDGS, respectively. Furthermore, the contents of total TriFAs were 5.7 and 6.3 times higher in corn DDGS than in wheat and mixed-cereal DDGS, respectively. In addition, both corn grains and corresponding DDGS had similar profiles of individual DFAs and TriFAs, indicating that ferulic acid cross-links in the corn cell wall are presumably not modified during fermentation and DDGS processing.


Assuntos
Ácidos Cumáricos/química , Triticum/química , Zea mays/química , Cromatografia Líquida de Alta Pressão , Dimerização , Destilação , Solubilidade
5.
Biochem J ; 462(3): 441-52, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24947135

RESUMO

Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 Å) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH ~6 and 66°C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller ß-sandwich domain. The active site contains an aspartate residue for acid-base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.


Assuntos
Amidoidrolases/metabolismo , Proteínas Fúngicas/metabolismo , Ocratoxinas/metabolismo , Amidoidrolases/isolamento & purificação , Aspergillus niger/enzimologia , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Proteínas Fúngicas/isolamento & purificação , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Conformação Proteica , Especificidade por Substrato
6.
J Biol Chem ; 288(37): 26764-74, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23902768

RESUMO

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (ß/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.


Assuntos
Glicosídeo Hidrolases/química , Polissacarídeo-Liase/química , Alga Marinha/enzimologia , Acarbose/química , Catálise , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Glucanos/química , Ácido Glutâmico/química , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Prótons , Sulfolobus solfataricus/enzimologia , Trissacarídeos/química , Valina/química , alfa-Glucosidases/metabolismo
7.
J Biol Chem ; 287(27): 22441-4, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22613728

RESUMO

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.


Assuntos
Frutose/análogos & derivados , Glucose/biossíntese , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo , Animais , Aspergillus niger/enzimologia , Abelhas/enzimologia , Catálise , Ativação Enzimática/fisiologia , Etanol/química , Fagopyrum/enzimologia , Frutose/química , Frutose/metabolismo , Glucose/metabolismo , Rodófitas/enzimologia , Solventes/química , Amido/metabolismo , Streptococcus mutans/enzimologia , Relação Estrutura-Atividade , Especificidade por Substrato , Água/química
8.
J Mol Biol ; 417(4): 279-93, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22330145

RESUMO

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed ß-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn(2+) and Mg(2+) located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn(2)(+)-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn(2+) in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn(2+) acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.


Assuntos
Frutose/análogos & derivados , Hidroliases/química , Phanerochaete/enzimologia , Zinco/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Frutose/química , Cetoses/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína
9.
Anal Biochem ; 410(2): 177-84, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21050837

RESUMO

Phytase (EC 3.1.3.-) hydrolyzes phytate (IP(6)) present in cereals and grains to release inorganic phosphate (P(i)), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P(i) liberated from IP(6). This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP(6)-lysozyme as a substrate. The assay is based on the principle that IP(6) forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP(6) in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P(i) from IP(6). This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline ß-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl(2), and phosphate, showed a concentration-dependent interference.


Assuntos
6-Fitase/química , 6-Fitase/metabolismo , Muramidase/química , Fosfatos/química , Ácido Fítico/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Muramidase/metabolismo , Fosfatos/metabolismo , Ácido Fítico/metabolismo , Especificidade por Substrato , Temperatura Ambiente
10.
BMC Endocr Disord ; 10: 17, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20958989

RESUMO

1,5-Anhydro-D-fructose (AF) is a mono-saccharide directly formed from starch and glycogen by the action of α-1,4-glucan lyase (EC 4.2.2.13). Our previous study has indicated that AF increases glucose tolerance and insulin secretion in NMRI mice after administration through a gastric gavage in a single dose at 150 mg per mouse. In this study, we used high-fat feeding of C57BL/6J mice to examine the influence of long-term administration of AF on glucose-stimulated insulin secretion in vivo and in vitro. We found that 8-weeks of high-fat feeding increased body weight, fasting blood glucose and insulin levels in C57BL/6J mice when compared to mice fed normal diet. Impaired glucose tolerance was also observed in mice receiving 8-weeks of high-fat diet. In contrast, AF (1.5 g/kg/day), administered through drinking water for 8-weeks, did not affect body weight or food and water intake in mice fed either the high-fat or normal diet. There was no difference in basal blood glucose or insulin levels between AF-treated and control group. Oral glucose tolerance test (OGTT) showed that AF did not affect glucose-stimulated insulin secretion in mice. In in vitro studies with isolated islets, AF did not influence glucose-stimulated insulin secretion in mice receiving either high-fat or normal diet. We therefore conclude that when given through drinking water for 8 weeks at 1.5 g/kg/day, AF has no effect on glucose-stimulated insulin secretion in C57BL/6J mice challenged with a high-fat diet.

11.
Planta Med ; 76(15): 1635-41, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20645241

RESUMO

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.


Assuntos
Frutose/análogos & derivados , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Metabolismo dos Carboidratos , Cárie Dentária/prevenção & controle , Frutose/química , Frutose/metabolismo , Frutose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Cetoses/metabolismo , Cetoses/farmacologia , Camundongos , Substâncias Protetoras/química , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Ratos
12.
Carbohydr Res ; 345(2): 181-90, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20004890

RESUMO

1,5-Anhydro-D-fructose (1,5AnFru) is a monoketosaccharide that can be prepared enzymatically from starch by alpha-1,4-glucan lyase or chemically from D-glucose or D-fructose in a few steps with high yields. The formed 1,5AnFru can be derivatized both enzymatically and chemically to interesting new carbohydrate derivatives, some with biological activities. For example dehydratases, isomerases and reductases can convert 1,5AnFru to enolones (as Ascopyrone P) and sugar alcohols with antimicrobial and antioxidant properties, while chemical modifications can give similar compounds as well as natural products like 1-deoxymannonojirimycin and Clavulazine. 1,5AnFru disaccharides (glycosyl 1-->4 1,5AnFru) have been prepared as well as glycosyl 1-->4 1,5-anhydro-D-tagatose.


Assuntos
Biocatálise , Frutose/análogos & derivados , Animais , Biotecnologia , Enzimas/metabolismo , Frutose/síntese química , Frutose/química , Frutose/metabolismo , Humanos
13.
Guang Pu Xue Yu Guang Pu Fen Xi ; 28(8): 1745-8, 2008 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-18975794

RESUMO

In the present paper, the authors study the photolumimescence spectra of the novel 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine casting film and vacuum-deposited film. Photolumimescence spectras of casting film on the quartz substrate were measured at 10, 77, 177 and 300 K, and the photolumimescence spectra of vacuum-deposited film with a thickness of about 200 nm on the silicon substrate was studied at room temperature (300 K). For 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine, the casting films all show fluorescence peaks at 942, 937, 942 and 942 nm and phosphorescence peaks at 1114, 1057, 1114 and 1114 nm in the photolumimescence spectra at 10, 77, 177 and 300 K, respectively. In the cases of 2,3-tetra-(2-isopropyl-5-methyl -benzoyl) hydrogen phthalocyanine, the peaks of excimers, which are related with the resistance ability of molecular aggregation, were found around 1673 nm as observed from photolumimescence spectra of the novel phthalocyanine casting films at 177 and 300 K. And the peak of excimers at 300 K is stronger than at 177 K also as can be seen from photolumimescence spectra of its casting films. With the increase in the temperature, the fluorescence peak was weakened and the peaks of excimers became stronger from the photoluminescence spectra of 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine casting films at 10, 77, 177 and 300 K. At the same time, the authors discussed the reason for coming into being 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine excimers as can be concluded from the structure of 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine molecules through the parameters of Chem 3D Ultra 9.0 MM2 calculation and simulated diagram of C4h isomer of 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine. The peaks of casting film and vacuum-deposited film of 2,3-tetra-(2-isopropyl-5-methyl -benzoyl) hydrogen phthalocyanine presented different maximum emission wavelength and full width at half maximum. The peak of 2,3-tetra-(2-isopropyl-5-methyl-benzoyl) hydrogen phthalocyanine vacuum-deposited films displays the maximum emission wavelengths around 1140 nm, while the maximum emission wavelengths of casting films show obvious differences compared with the vacuum-deposited films. The usual full width at half maximum is approximately 300 nm for casting film, which is in contrasts with that the full width at half maximum is about 100 nm for the vacuum-deposited film as can be seen from photolumimescence spectra of 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine casting film and photolumimescence spectra of 2,3-tetra-(2-isopropyl-5-methylbenzoyl) hydrogen phthalocyanine vacuum-deposited film.

14.
IUBMB Life ; 60(12): 798-809, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18785261

RESUMO

In many living forms, glucose is stored in the polymeric form of glycogen and starch as a source of carbon and energy. Upon need, these polymers are broken down to their building block glucose in the presence of water and hydrolases, and glucose 1-phosphate in the presence of phosphate and phosphorylase. In the last decade, we established an alternative glycogen catabolic pathway, the so-called Anhydrofructose (AF) pathway, the description of which was approved by IUBMB in 2006. It is a pathway on the formation of an array of secondary metabolites from glycogen via the central intermediate 1,5-anhydro-D-fructose (AF). Furthermore, we demonstrated the occurrence of this pathway in both eukaryota and prokaryota. Metabolites of this pathway have been identified in mammals including humans. In this review, the physiological and molecular functions of these metabolites in the AF pathway as well as its regulation are discussed.


Assuntos
Frutose/análogos & derivados , Glicogênio/metabolismo , Transdução de Sinais , Frutose/metabolismo , Humanos
15.
Biochim Biophys Acta ; 1760(9): 1314-22, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16822618

RESUMO

Anhydrofructose (AF) pathway describes the catabolism of alpha-1,4-glucans of glycogen, starch and maltosaccharides to various metabolites via the central intermediate AF. The reaction sequence of the pathway consists of more than 10 enzymatic steps. This pathway occurs in certain bacteria, fungi, algae and mammals. In this communication, the AF pathway and its regulatory mechanisms in these organisms are presented and the metabolites of this pathway as antioxidants and antimicrobials in biotic and abiotic stress responses and in carbon starvation signaling are discussed.


Assuntos
Frutose/análogos & derivados , Frutose/metabolismo , Transdução de Sinais , Animais , Desidrogenases de Carboidrato/metabolismo , Catálise , Humanos
16.
Appl Environ Microbiol ; 72(2): 1248-57, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16461673

RESUMO

The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-D-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-D-mannitol (AM) and the further conversion of AM to D-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli. In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti, Brucella suis, and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.


Assuntos
Frutose/análogos & derivados , Sinorhizobium/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Carboidratos/análise , Carboidratos/biossíntese , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/genética , Frutose/metabolismo , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizobiaceae/enzimologia , Rhizobiaceae/genética , Homologia de Sequência de Aminoácidos , Sinorhizobium/enzimologia , Sinorhizobium/genética , Sinorhizobium/crescimento & desenvolvimento , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/genética
17.
Protein Expr Purif ; 46(1): 56-63, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16343940

RESUMO

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.


Assuntos
Hordeum/enzimologia , Pichia/enzimologia , alfa-Glucosidases/genética , Cromatografia de Afinidade , Primers do DNA , Fermentação , Cinética , Pichia/genética , Pichia/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Ultrafiltração , alfa-Glucosidases/isolamento & purificação , alfa-Glucosidases/metabolismo
18.
J Agric Food Chem ; 53(24): 9491-7, 2005 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-16302767

RESUMO

The anhydrofructose pathway describes the degradation of glycogen and starch to 1,5-anhydro-D-fructose (1,5AnFru) and its further conversion to the enolone ascopyrone P (APP) via the transit intermediate ascopyrone M. The two products, 1,5AnFru and APP, were examined in this study for their effects in controlling the browning of selected fruits, vegetables, and beverages. The results showed that 1,5AnFru had an antibrowning effect in green tea and was able to slow turbidity development in black currant wine. APP proved to be an antibrowning agent comparable to kojic acid. It showed an antibrowning effect in a range of agricultural products, such as various cultivars of apple, pear, potato, lettuce, and varieties of green tea in an efficacy concentration range from 300 to 500 ppm. Mechanism studies indicated that, like kojic acid, APP showed inhibition toward plant polyphenol oxidase and was able to decolor quinones.


Assuntos
Frutose/análogos & derivados , Glicogênio/metabolismo , Reação de Maillard/efeitos dos fármacos , Amido/metabolismo , Camellia sinensis/efeitos dos fármacos , Catecol Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Frutose/metabolismo , Frutose/farmacologia , Frutas , Verduras
19.
Drug Chem Toxicol ; 28(3): 263-72, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16051552

RESUMO

1,5-anhydro-d-fructose (1,5-AF) is a novel monosaccharide produced by the action of alpha-1,4-glucan lyase (EC 4.2.2.13) on glycogen, starch, or related substrates such as maltose and maltosaccharides. 1,5-AF is of interest as a compound to be used as a food supplement because of its antioxidant, antimicrobial, and antidiabetic properties. This enforces the safety of 1,5-AF and therefore, in the current study, four groups of male and female Sprague-Dawley rats were provided with 1,5-AF in the drinking water (at 0 or 1.0 g/kg body weight daily) for a period of 90 days (n=10 in each group). All the animals survived, and no clinical signs of toxicity or alterations in hematological or clinical chemistry parameters were observed. Furthermore, organ weight and histopathological examination of brain, heart, urinary bladder, gastrointestinal tract, and pancreas were normal after 1,5-AF treatment. Moreover, there was no change in food consumption, water intake, or body weight gain in rats receiving 1,5-AF. In conclusion, administration of 1,5-AF did not induce any significant toxicological effects and, therefore, 1,5-AF seems safe to administer in vivo over a long period of time.


Assuntos
Frutose/análogos & derivados , Animais , Contagem de Células Sanguíneas , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Frutose/química , Frutose/toxicidade , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais
20.
Biochim Biophys Acta ; 1723(1-3): 63-73, 2005 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-15716041

RESUMO

The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-D-fructose (1,5AnFru). Enzymes that form 1,5AnFru, ascopyrone P (APP), and ascopyrone M (APM) have been reported from our laboratory earlier. In the present study, APM formed from 1,5AnFru was found to be the intermediate to the antimicrobial microthecin. The microthecin forming enzyme from the fungus Phanerochaete chrysosporium proved to be aldos-2-ulose dehydratase (AUDH, EC 4.2.1.-), which was purified and characterized for its enzymatic and catalytic properties. The purified AUDH showing a molecular mass of 97.4 kDa on SDS-PAGE was partially sequenced. Total 332 amino acid residues in length were obtained, representing some 37% of the AUDH protein. The obtained amino acid sequences showed no homology to known proteins but to an unannotated DNA sequence in Scaffold 62 of the published genome of the fungus. The alignment revealed three introns of the identified AUDH gene (Audh; ph.chr), thus the first gene coding for a neutral sugar dehydratase is identified. AUDH was found to be a bi-functional enzyme, being able to dehydrate 1,5AnFru to APM and further isomerizing the APM formed to microthecin. The optimal pH for the formation of APM and microthecin was pH 5.8 and 6.8, respectively. AUDH showed 5 fold higher activity toward 1,5AnFru than toward its analogue glucosone, when tested at concentrations from 0.6 mM to 0.2 M. Based on the characteristic UV absorbance of microthecin (230 nm) and APM (262 nm) assay methods were developed for the microthecin forming enzymes.


Assuntos
Frutose/análogos & derivados , Frutose/metabolismo , Glicogênio/metabolismo , Hidroliases/fisiologia , Cetoses/biossíntese , Sequência de Aminoácidos , Catálise , Hidroliases/química , Hidroliases/genética , Hidroliases/isolamento & purificação , Dados de Sequência Molecular , Especificidade por Substrato , Terminologia como Assunto
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